The blood glucose level is an important marker for diabetes. The use of PQQGDH has already been commercialized as one method for measuring the glucose concentration.
PQQGDH is a glucose dehydrogenase that employs pyrroloquinoline quinone as a coenzyme, and it catalyzes the oxidation of glucose with the production of gluconolactone. PQQGDH is known to occur as a membrane-bound enzyme and as a water-soluble enzyme. Membrane-bound PQQGDHs are single peptide proteins with molecular weights of approximately 87 kDa and are widely encountered in various Gram-negative bacteria. Water-soluble PQQGDH, on the other hand, has been identified in several strains of Acinetobacter calcoaceticus, and its structural gene was cloned and its amino acid sequence was determined (GenBank accession number X15871; Mol. Gen. Genet. (1989), 217: 430-436). The results of X-ray crystal structural analysis of water-soluble PQQGDH from Acinetobacter calcoaceticus have been reported and the higher order structure of the enzyme, and most importantly the active center, has been elucidated. (A. Oubrie et al., J. Mol. Biol., 289, 319-333 (1999); A. Oubrie et al., The EMBO Journal, 18(19), 5187-5194 (1999); A. Oubrie et al., PNAS, 96(21), 11787-11791 (1999)). Water-soluble PQQGDH from Acinetobacter baumannii has also been identified (GenBank accession number E28183).
PQQGDHs have a high oxidation activity for glucose and do not require oxygen as an electron acceptor because they are coenzyme-linked enzymes. As a result they are expected to find application in glucose assays and particularly as the recognition element of glucose sensors. A problem with PQQGDHs, however, is their low selectivity for glucose. In particular, PQQGDH also has a high activity for maltose, and thus accurate assay is difficult in patients receiving a maltose-containing infusion solution. In this case, the apparent blood sugar level will be higher than the actual blood sugar level, which could lead to a risk of hypoglycemia caused by administering insulin to the patient based on the measured level. Accordingly, a PQQGDH that exhibits a higher selectivity for glucose versus maltose is desired for the enzyme used for measurement of the blood sugar level.
The present inventor has already reported several modified PQQGDHs that exhibit an increased selectivity for glucose (for example, WO 00/66744, Japanese Patent Application Laid-open No. 2001-346587, and WO 2004/005499), but a modified PQQGDH that exhibits an even higher selectivity and/or an even higher enzymatic activity is still required.
The reference documents cited herein are listed below. The contents of these documents are hereby incorporated by reference in its entirety. None of these documents are admitted to constitute a prior art of the present invention.    Patent Document 1: WO 00/66744    Patent Document 2: Japanese Patent Application Laid-open No. 2001-346587    Patent Document 3: WO 2004/005499    Nonpatent Document 1: Mol. Gen. Genet. (1989), 217:430-436    Nonpatent Document 2: A. Oubrie et al. (1999) J. Mol. Biol., 289, 319-333    Nonpatent Document 3: A. Oubrie et al. (1999) The EMBO Journal, 18(19), 5187-5194    Nonpatent Document 4: A. Oubrie et al. (1999) PNAS, 96(21), 11787-11791